With the increased use of engineered nanoparticles in medicine (diagnostics and therapeutics), electronics, cosmetics, and textiles and the corresponding awareness of the toxicity from such nanoparticles, there is a critical need to develop methods to evaluate their toxicity, especially at low concentrations that cause oxidative stress and adverse long-term health effects. Macrophages are the primary defense cells in the lung responsible for uptake, degradation and clearance of foreign particles, all biophysical processes which rely upon an intact cytoskeleton. Impairment of these biophysical processes due to nanoparticle-mediated cytoskeletal oxidation contributes to toxicity via reduced particulate clearance and inflammation. This inflammation and associated overproduction of reactive oxygen species further oxidize RNA/DNA/proteins, leading to fibrosis, mutagenesis and lung cancer. To study how macrophage uptake and transport are affected by toxic nanoparticles, we have developed a novel technique to track intracellular transport of phagocytosed magnetically modulated optical nanoprobes (MagMOONs). These MagMOONs are micron sized tracer particles with one hemisphere coated by gold creating an orientation-dependent scattering and fluorescence signal. Tracking rotational transport via intensity changes allows analysis of many particles simultaneously even at low magnification (e.g. endoscopy). Results from a related technique pioneered by Dr. Miller (Co-I) demonstrate that intracellular rotation rate is a powerful indicator for macrophage health in vivo, and that rotatio is impaired by toxic nanoparticles in vitro. However, Dr. Miller's technique measures only the average transport-mediated rotational diffusion rate from ensembles of millions of cells, and is unable to localize particles or observe motion of individual particles and cells. The objective of this proposal is to extend the biophysical rotation methods to single particles and localized regions via optical tracking of the MagMOONs. Single particle studies will provide a more detailed mechanistic model for intracellular transport and NP- induced cytoskeletal oxidation. Our central hypothesis is that differences in particulate matter composition affect intracellular phagosome transport via ROS generation. These local cytoskeletal oxidations, in concert with the oxidation of secondary messengers and the depletion of antioxidants, cause global cytoskeletal damage and dysfunction, DNA damage, and cell death. We will test this hypothesis by studying the effect of nanoparticle composition on the motion and transport of single magnetic tracer particles in macrophages. We will also use our assay to study the protective effects of ROS scavengers such as NAC. By optically tracking MagMOONs in tissue phantoms we can also show feasibility for eventual in vivo detection in animal models with fewer tracer particles and localization of the particles in the lungs through endoscopy and transdermal X- ray excited optical luminescence (XEOL) imaging. The proposed research is significant because the technique developed in this project will have important applications for detecting and understanding nano-toxicity. PUBLIC HEALTH RELEVANCE: This research project develops a novel biophysical tool for evaluating the toxicity of engineered and environmental nanoparticles within individual cells. The proposed research is relevant to public health because we develop a novel bioanalytical imaging technique to measure nanoparticle mediated cytoskeletal dysfunction and damage (toxicity) on immune system cells. Specifically, we measure toxicity of copper oxide and titanium dioxide which are widely used and have important ramifications for human health.